Project Summary/Abstract The ability of microorganisms to form biofilms is a critical virulence factor for infection of the human host. Due to their complex structural make up, including multi-layer cell communities and a prominent extracellular matrix, biofilms are often recalcitrant to antimicrobial treatment. The opportunistic fungal pathogen Candida albicans is one of the leading causes of nosocomial infections due, in large part, to its ability to form complex biofilms. C. albicans can colonize every major organ in the body and forms biofilms on both medical implants and mucosal surfaces. Furthermore, in oropharyngeal candidiasis (OPC), the most common fungal infection in humans, C. albicans biofilms are formed on oral tissues and dentures. From the oral cavity, C. albicans can disperse to seed new infection sites and invasive candidiasis (IC) in the bloodstream. Mortality rates for IC remain at ~40% despite clinical intervention, with most cases thought to originate from commensal niches. Dissecting the molecular mechanisms dictating C. albicans colonization of the oral niche is therefore crucial in order to better treat chronic infections and to also minimize disease progression to IC. A well-defined transcriptional regulatory network (TRN) consisting of 9 master transcription factors (TFs) controls biofilm development in C. albicans. Multiple TFs in this network bind to their own regulatory regions and to those of the other 8 factors, and it is postulated that biofilm regulation depends on physical interactions between these TFs. Interestingly, sequence analysis indicates that 7 TFs in the circuit contain prion-like domains (PrLDs). These are intrinsically disordered regions and recent studies indicate that they can enable proteins to undergo a phenomenon known as phase separation or liquid-liquid demixing. The separation of proteins into a more concentrated and a more dilute phase is implicated in the formation of membrane-less organelles and in diseases such as Alzheimer?s. The experiments proposed in this application, however, are the first to examine how TFs can form phase-separated condensates to regulate biofilm formation in C. albicans. In my preliminary data, I show that five TFs from the biofilm network have been purified and several of these proteins readily undergo phase separation to form liquid-like condensates in vitro. Importantly, upon overexpression in a mammalian cell reporter system, I have also found that TFs can form PrLD-mediated condensates in living cells. Moreover, C. albicans strains expressing biofilm TFs with PrLD deletions are unable to form mature biofilms. Based on this data, I hypothesize that master biofilm TFs undergo phase separation to regulate target genes in the biofilm network, and that disruption of this process will block biofilm formation in the oral cavity. In Aim 1, I will determine if PrLDs mediate phase separation of multiple biofilm TFs in vitro. In Aim 2, I will define the role of PrLD-containing TFs in promoting C. albicans biofilm formation and virulence in an OPC model. These findings will inform how TF complexes regulate gene expression in eukaryotes, and will lead to therapeutic strategies for combating fungal biofilm formation and pathogenesis.